Cryopreservation plays a pivotal role in the ability to preserve the long term viability, structure and function of biological cells and tissues. Cryopreservation has long played a role in the area of human assisted reproduction. The ability to successfully store and bank human gametes is essential in the safe and efficient treatment of human infertility. We are developing a novel method of human oocyte cryopreservation using microcapillary freezing. Conventional cryopreservation techniques leave large mammalian cells susceptible to intracellular damage secondary to the formation of intracellular ice crystals. The alternative to conventional slow freezing is the process of vitrification, which relies on very high rates of cooling to freeze cells in a glassy state. The problem with current vitrification protocols is that they require extremely high concentrations (generally 6-8M) of cryoprotectant agents (CPA's). Such high CPA levels are themselves toxic to cells. The ideal cryopreservation protocol, then, would be one that combines the benefits of conventional slow freezing (i.e., reduced toxicity secondary to low CPA levels) with the benefits of vitrification (i.e., resistance to intracellular ice crystal formation). Our microcapillary freezing technique seeks to bridge this gap. We have shown that the process of vitrification can be optimized by maximizing the rate at which the sample is cooled. This allows for the use of lower CPA concentrations. The cooling rate needed to achieve "ice-free" conditions directly determines the required level of CPA. The higher the rate of cooling, the lower the necessary CPA concentration needed for vitrification. Our approach minimizes the thermal mass of the freezing vessel by using microcapillaries made of highly conductive quartz. We already have demonstrated that we can achieve cooling rates in excess of 200,000[unreadable]C/min and can vitrify 2M CPA solutions. We now intend to adapt this groundbreaking new knowledge to help solve the vital clinical problem of human oocyte cryopreservation. In Specific Aim 1, we will develop a robust, simple, easy to use, microfluidics quartz capillary freezing device, for the handling and manipulation of oocytes for ultrarapid vitrification. In Specific Aim 2, we will validate the use of the novel microfulidics quartz capillary freezing device for ultrarapid vitrification using mouse oocytes. In Specific Aim 3, we will validate the use of the novel microfulidics quartz capillary freezing device for ultrarapid vitrification using fail to fertilize human oocytes. PUBLIC HEALTH RELEVANCE: Cryopreservation plays a pivotal role in the ability to preserve the long term viability, structure and function of biological cells and tissues. Oocyte cryopreservation is one of the most sought after advances in assisted reproductive technologies. The applications of oocyte cryopreservation include: the preservation of future fertility in women at risk for losing their reproductive functions due to cancer treatment, the avoidance of the ethical and legal dilemmas surrounding the cryopreservation of human embryos, as well as for women delaying initiating pregnancy. We are developing a novel method of human oocyte cryopreservation using a micro capillary freezing device. This novel technology combines the benefits of traditional slow freezing protocols (low concentrations of cryoprotectant) with the benefits of vitrification (resistance to intracellular ice crystal formation). We now intend to adapt this groundbreaking new knowledge to help solve the vital clinical problem of human oocyte cryopreservation.